In the United States there has been an increase in tick-borne infections over the last decade. Among the most threatening of these are the rickettsial infections. The best known of the human rickettsial infections is responsible for Rocky Mountain Spotted Fever (RMSF), a disease that has remained severe with a fatality rate of 3-10%, even with modern medical care. The etiologic agent of RMSF, Rickettsia rickettsii, is one of 18 recognized members of the Rickettsia Spotted Fever Group (SFG) and a NIAID Category C Priority Pathogen. Tick infection rates for R. rickettsii are believed to be approximately 1-3% where RMSF is endemic. In addition, many other potentially pathogenic bacteria circulate and are maintained in these tick populations with infection rates in some studies as high as 36%. Despite non-specific detection using molecular and visual methods, the majority of these organisms have eluded accurate identification. Specific infection rates and the potential human pathogens with which these ticks are infected, are poorly documented, but could include several Rickettsia (NIAID Category C Priority Pathogens) and Rickettsia-like organisms (categorization unknown). Accurate identification of many of these agents has remained elusive, likely due to poor conservation of known molecular targets to a very limited amount of "unknown" genetic material that is routinely recovered from field-collected ticks. This project will focus on optimization of methods for culturing of Rickettsia and Rickettsia-like organisms from field-collected Amblyomma americanum ticks, such that they can be characterized and identified using existing techniques. This target species was selected for this Small Grant proposal for proof of principle because these ticks are common in Maryland, have low infection rates with known human pathogens in Maryland, but have relatively high infection rates with identifiable non-pathogenic Rickettsia and Rickettsia-like organisms that have evaded identification. Knowledge of what agents are components of the natural ecological background is integral to the accurate assessment of the introduction and spread of native and non-native pathogens, either through incidental or more malicious means. [PARAGRAPH] Hypotheses: Shell-vial inoculation will result in isolation of known and novel Rickettsial infections from Maryland ticks and purification and molecular analysis of these isolates can lead to positive identification of these organisms. [PARAGRAPH] Specific aims of this proposal are to: [PARAGRAPH] 1. Use shell-vial culture techniques to culture/isolate known and unknown Rickettsia-like organisms from man-biting ticks of Maryland. We will isolate and culture Rickettsia-like organisms from tick hemolymph in mammalian and arthropod cell lines that have been previously shown to be permissive to infection with diverse Rickettsial species. [PARAGRAPH] 2. Identify and characterize Rickettsia and Rickettsia-like organisms recovered in Aim #1. Preliminary characterization of the Rickettsia and Rickettsia-like organisms will involve differentiation between Spotted Fever Group and non-Spotted Fever Group Rickettsia from A. americanum. Subsequent identification will involve sequencing of the citrate synthase and other target genes for molecular identifications and phylogenetic reconstructions. PUBLIC HEALTH RELEVANCE: The primary goal of this project is to use shell-vial culture techniques to culture/isolate known and unknown Rickettsia and Rickettsia-like organisms from man-biting ticks of Maryland. Additionally, we will utilize the genetic material recovered via culture, to develop, optimize and utilize molecular methods to characterize these novel organisms that have eluded identification with existing tools.